Barclays boss Antony Jenkins has told the bank's 140,000 employees to sign up to a new code of conduct, or leave.
The demand has come as employees prepare to learn what bonuses they will receive for their services in 2012.
He said that performance would be assessed "not just on what we deliver, but on how we deliver it".
Mr Jenkins replaced Bob Diamond who quit over Barclays' role in rigging the Libor rate used in trillions of pounds of financial contracts.
Barclays was just the first of several international banks to be implicated in the scandal, and was fined a total £290m by US and UK regulators.
The bank, along with most of the other major UK High Street lenders, has also been found culpable in recent years of mis-selling unnecessary payment protection insurance to mortgage borrowers, and overly complex, over-priced interest rate and currency hedges to small businesses.
'Rules have changed'
Staff have been told to sign up to five key values - respect, integrity, service, excellence and stewardship.
Bonuses would be assessed against the new "Purpose and Values" criteria.
"There might be some of you who don't feel they can fully buy in to an approach which so squarely links performance to the upholding of our values," said Mr Jenkins.
"My message to those people is simple: Barclays is not the place for you. The rules have changed. You won't feel comfortable at Barclays and, to be frank, we won't feel comfortable with you as colleagues."
He attacked the bank's increasingly aggressive work culture of the previous 20 years, which, he said, focused too much on making a quick buck instead of upholding the values and long-term reputation of the bank.
An early indication of the new boss's attitude came with his appointment last year of Hector Sants, former boss of the UK's banking watchdog, the Financial Service Authority, to head the Barclays' compliance department.
It is unclear how the size of remuneration packages at Barclays will be affected by the new focus on ethics.
The total size of Barclays' bonus pool for last year will not be disclosed until next month. Two US banks - Goldman Sachs and JP Morgan - controversially announced this week that they had decided to increase their pots by 6% and 5% respectively this year.
Mr Jenkins is also due to unveil next month a new business strategy for Barclays, which he said would "excite" the workforce.